POS0141 PLATELET-SELECTIN PRIME LUPUS NEUTROPHILS TO PRODUCE MITOCHONDRIAL ROS AND PARTICIPATE IN SLE PATHOGENESIS
Authors: M. Scherlinger, V. Guillotin, P. Vacher, I. Douchet, E. Lazaro, C. Richez, P. Blanco
Journal: Annals of the Rheumatic Diseases (2022)

<h3>Background</h3> In patients with active systemic lupus erythematosus (SLE), circulating platelets have an activated phenotype characterized by the expression of P-selectin (CD62P). We have shown that in human SLE, platelets interact with T regulatory cells and repress their immunosuppressive functions through a P-selectin/CD15s-dependent interaction (1). Preliminary results showed that neutrophils express high level of CD15s, predicting a possible platelet/neutrophil interaction in SLE. <h3>Objectives</h3> Investigate platelet/neutrophils interaction in human SLE and evaluate its impact on neutrophil functions, and lupus pathogenesis. <h3>Methods</h3> Patients with SLE responding to the 2019 ACR/EULAR criteria were recruited for blood sampling (n = 30). Disease activity was measured using SLE disease activity index (SLEDAI-2K), and patients were considered active if SLEDAI-2K ≥ 6. Platelet-neutrophils aggregates were identified as (platelet) CD61<sup>+</sup> (neutrophil) CD66b<sup>+</sup> cells using flow cytometry on fresh blood samples. Single-cell cytosolic calcium and ROS imaging was performed by incubating cell with either a fluorescent calcium dye (cali-520), or a mitochondrial specific dye (MitoSox). Coverslips were mounted in an Attofluor cell chamber positioned on the stage of an inverted epifluorescence microscope (Olympus, IX70). Mitochondrial polarization of human neutrophil was evaluated by incubating cells with TMRM (100 nM) for 30 minutes. Platelet-free plasma was isolated by two sequential centrifugations (3500xg) of EDTA-anticoagulated blood and stored for subsequent evaluation of soluble P-selectin (using ELISA) and (platelet-derived) microparticular P-selectin (using flow cytometry). <h3>Results</h3> In healthy donors (HD) and patients with SLE, circulating neutrophils expressed significantly higher levels of the P-selectin ligand CD15s compared to lymphoid subsets (p &lt; 0.001), predicting platelet/neutrophil interactions. In contrast to HD and patients with inactive SLE, those with active disease had a significant increase of circulating platelet-neutrophils aggregates (p &lt; 0.05), and these aggregates correlated with the SLEDAI (r = 0.59, p &lt; 0.001). The incubation of human neutrophils with recombinant P-selectin induced a strong intracellular calcium signaling which was inhibited by preincubating neutrophil with anti-PSGL1 antibody (blocking P-selectin/CD15s interaction) or with a Syk kinase inhibitor. Similarly, P-selectin induced a mitochondrial ROS release in a CD15s- and Syk-dependent manner. Interestingly, incubation of neutrophils with anti-dsDNA IgG and P-selectin induced mitochondrial depolarization, which was absent with either stimulus alone. Soluble and platelet-derived microparticular P-selectin levels were significantly increased in patients with active SLE compared to inactive patients or healthy donors (p &lt; 0.05 and p &lt; 0.001, respectively). In a longitudinal analysis of SLE patients, soluble and microparticular P-selectin levels closely followed clinical (SLEDAI) and biological (C3 levels) markers of SLE disease activity. <h3>Conclusion</h3> P-selectin levels are increased in active SLE and follow hallmark of disease activity. P-selectin induces calcium/mitochondrial ROS signaling in lupus neutrophils which are key players in SLE pathogenesis. We hypothesize that the inhibition of P-selectin pathway might be a promising target in SLE. <h3>References</h3> [1]Scherlinger M. <i>et al.</i> (2021), <i>Science Translational Medicine</i>, 13(600):eabi4994. <h3>Disclosure of Interests</h3> None declared